Kit for a Determination Method for Calprotectin and the Use of Calprotectin as a Predictive Marker for Cardiovascular Disease

ABSTRACT

An assay kit for the use in a method for the detection of potential for CVD or propensity to CVD in a human or non-human animal subject. The method including assessing the concentration of calprotectin in a calprotectin-containing sample taken from the subject.

The present invention relates to an assay method for detecting potentialfor or propensity to cardiovascular disease (CVD) in a subject, e.g. ahuman or non-human animal, especially a mammal, and in particular to anassay method which may be used to detect a potential for CVD or apropensity to CVD before the onset of CVD symptoms noticeable by thesubject.

CVD is a major source of ill health among the human population. In 1998approximately 40% of all deaths in the western world was a result of CVD(i.e. 1 in every 2.5 deaths). For 2002, it is estimated that, in theUSA, over one million people will suffer from a new or recurrentcoronary attack, and more than 40% of the people suffering from theseattacks will die. Many of these people will die suddenly without everhaving been hospitalised or treated. Many will not have realised thatthey were susceptible to CVD.

Early or pre-emptive treatment such as a change of diet, reduction orcessation of smoking, increase in exercise, reduction of body weight,etc., has, however, a high success rate of preventing CVD or reducingthe propensity to CVD. Thus if CVD or potential for or propensity to CVDcan be detected, effective treatment is available.

There is accordingly a need for methods which can be used to detect CVDand especially, the potential for or propensity to CVD, before thedisease has progressed beyond the stage where treatment (e.g. change oflife style and/or habit) is routinely successful. In particular, thereis a need for methods which can be used to detect CVD at the earlystages when the symptoms are not apparent to a subject or to a thirdparty, e.g. a physician, i.e. methods for testing “symptom-free”subjects are required.

Such methods may be used to screen the general population (i.e. in massscreening) or at-risk groups within the population, e.g. males over 40,workers in high stress jobs, individuals with unhealthy diets,individuals suffering from clinical obesity, smokers, etc. In caseswhere potential for CVD or propensity to CVD is diagnosed, pre-emptivetreatment may be given and/or the patient may be encouraged to makeadjustments to lifestyle and habit.

Likewise, where potential for CVD or propensity to CVD is detected, apatient may be submitted to further testing, e.g. using more expensiveor time consuming techniques, such as ECG, with and without physicalactivity, radioisotope imaging of myocardial perfusion, X-ray (e.g. CT)myocardial angiography, MR myocardial angiography or perfusion imaging,etc. Thus by confirming the possible presence of, or potential for, orpropensity to, CVD by using the cheap and facile assay method of theinvention in an initial screen (e.g. in a mass screen of “symptom-free”healthy subjects) the likelihood of detecting or identifyingundiscovered CVD, or potential for CVD, before health damage becomesirreversible is increased whilst, at the same time, unnecessary use ofexpensive and time-consuming tests is limited.

By “CVD” is meant any condition of the heart, arteries, or veins whichdisrupts the supply of oxygen to life-sustaining areas of the body suchas the brain, the heart etc. Examples of CVDs are arteriosclerosis,acute myocardial infarction, angina pectoris, ischemic heart disease,cerebrovascular disease, stroke, subarachnoid haemorrhage,intra-cerebral haemorrhage, cerebral infarction, congestive heartfailure, angina, heart attack, cardiac arrest and arrhythmia.

The present invention is based on the surprising finding that theprotein calprotectin is a useful “marker” or “indicator” of potentialfor CVD or propensity to CVD before the onset of CVD symptoms (i.e. insymptom-free subjects). In particular it has been surprisingly foundthat abnormally high calprotectin levels in various body fluids isindicative of susceptibility to CVD before the onset of CVD symptoms isapparent to a subject or to a third party (e.g. a physician).

For the avoidance of doubt, the term calprotectin is used hereinsynonymously with “L1 protein”, “MRP 8/14”, “cystic fibrosis(associated) antigen (CFA)” and “calgranulin”.

Calprotectin exists in both dimeric and trimeric forms. As a dimer,calprotectin comprises the polypeptide chains S100A8 and S100A9. As atrimer, calprotectin is a 36 kDa heterotrimeric protein with two heavy(14 kD) and one light chain (8 kD) non-covalently linked.

Calprotectin is a calcium binding protein and when bound to calcium,calprotectin is resistant to heat and to proteolysis. This may allow fora wide range of assay techniques and conditions to be employed.

Epitope mapping of calprotectin shows that antibodies with specificityfor the complex and/or its single protein chains may be produced. Atleast four separate immunogenic sites have been shown to exist on thecalprotectin complex. Some antibodies recognise either the heavy or thelight chain, whilst others recognise both.

Calprotectin is found in cells, tissues and fluids in all parts of thehuman body and is derived predominantly from neutrophils and monocytes.Calprotectin is probably present in all individuals since amongst morethan 5,000 individuals tested, no calprotectin free individual wasfound. Calprotectin is also found in rats, mice, rabbits, sheep, cattleand pigs. It is therefore an abundant ubiquitous molecule.

In vivo, calprotectin is involved in numerous biological functionsincluding intracellular signal transduction, neutrophil activation,inhibition of intracellular enzymes involved in cell proliferation,antimicrobial activity and in neutrophil defence. Calprotectin is also aregulatory protein in inflammatory reactions and in this role mayfunction to stimulate immunoglobulin production, chemotactic factoractivity and neutrophil immobilising factor.

Whilst body fluids probably always comprise calprotectin, theconcentration of calprotectin in various body fluids has been found tochange, for example, to increase, in a number of disease conditions(e.g. inflammatory, infectious and malignant diseases). Thus measurementof the concentration of calprotectin in body fluid from patientssuffering from such disease conditions (i.e. in individuals showingsymptoms noticeable to the subject and/or to a third party) andcomparing the calprotectin concentration determined to that in bodyfluid from, for example, a healthy (i.e. a non-diseased) subject may beused as a means of diagnosing such diseases.

For example, whilst the symptoms of bacterial and viral infections arevery similar and diagnosis from their symptoms alone may be difficult,the concentration of calprotectin in the plasma/serum of the infectedsubject increases approximately 1 to 2 times with viral infections butaround 1 to 18 times with bacterial infections. Thus the subject havingnoticed the symptoms of infection, can have the concentration ofcalprotectin in their body fluid measured and their infection diagnosedand treated accordingly.

Other diseases in which calprotectin may be used as a diagnostic testinclude: rheumatic diseases (e.g. rheumatoid arthritis, juvenilerheumatoid arthritis, systemic lupus erythematosus), Sjøgrens syndrome,intraocular inflammatory conditions, cystic fibrosis, acute and chroniclung disease, lung carcinoma (squamous cells), pulmonary cancers,colorectal cancer, inflammatory bowel disease, gastric cancer,colorectal adenoma or cancer, Chrohn's disease, ulcerative colitis,gastrointestinal mucosal inflammation, urinary stones, alcoholic liverdisease, oral inflammatory mucosal disease, CNS inflammatory disease(e.g. multiple sclerosis and acute encephalitis), HIV infection,secondary CNS infections in HIV infected patients, urinary tractinfections, cystitis, pyelonephritis, endogenous posterior uveitis,haematological conditions (e.g. leukaemia), febrile conditions(infectious and non-infectious), acute myocardial infarction andapheresis.

The plasma concentration of calprotectin has also been found to increaseduring open heart surgery (Semb, A. G. et al, Eur. J. Cardio-thoracSurg. (1991) 5:363-367, Saatvedt, K. et al., Scand. J. Thor. Cardiovasc.Surg. (1996) 30: 53-60, Moen, O. et al., Perfusion (1994) 9:109-117).More specifically, Saatvedt et al. report that calprotectinconcentration rises after the start of cardiopulmonary bypass and peaks48 hours post-operatively.

It has now surprisingly been found that the potential for CVD orpropensity to CVD in a subject can be assessed by determining theconcentration of calprotectin in a calprotectin-containing sample takenfrom said subject. In other words, it has been found that determinationof the concentration of calprotectin in a calprotectin-containing sampletaken from a subject can be used to predict, prior to the onset ofsymptoms which are noticeable to the subject or to a third party (e.g. aphysician) whether or how likely the subject is to suffer CVD.

By “potential for” or “propensity to” is meant the likelihood orprobability that the currently symptom-free subject being tested willsuffer CVD in the future. This might take the form of an index, ratio,percentage or similar number reflective of the relative risk of CVD inthe future (e.g. in the following 1-2 years, at least in the following 6months).

Thus viewed from one aspect the invention provides an assay method forthe detection of potential for CVD or propensity to CVD in a human ornon-human animal subject, said method comprising assessing theconcentration of calprotectin in a calprotectin-containing sample takenfrom said subject, e.g. a sample of blood, plasma, serum, cerebrospinalfluid, oral fluid, urine, faeces, synovial or empyema fluid.

By “assessing” it is meant that a quantitative or semi-quantitativevalue for the concentration of calprotectin is determined. This may bethe value for the concentration of the sample as tested, e.g. aftertreatment to remove the cells or other sample components not beingassayed for, or to concentrate or dilute the sample or to transfer thecalprotectin to a separate medium, e.g. solid substrate.

Alternatively, the assessment may simply be qualitative, i.e. toindicate whether the calprotectin is above or below one or morepre-selected threshold values, e.g. values indicative of absence ofpotential for CVD or propensity to CVD as detectable by the assay. Theprecise values for these threshold values or other reference values forcalprotectin may depend on the nature of the sample, the age, weight,sex and species of a subject and may be determined in a routine mannerby measuring the calprotectin concentration of the relevant body fluidof equivalent subjects without CVD or with CVD at various stages ofdevelopment.

A value indicative of calprotectin concentration determined or“assessed” in accordance with the method of the invention may be anabsolute concentration of calprotectin or may alternatively be an index,ratio, percentage or similar number reflective of the concentration ofcalprotectin.

A body sample used in the assay method of the invention may be anycalprotectin-containing sample, e.g. a body fluid or tissue sample, or asuspension etc. Preferably, the sample will be a body fluid, e.g. urine,cerebrospinal fluid, oral fluid, synovial fluid or empyema fluid, ormore preferably, blood or a blood derived sample. When this is the case(i.e. when blood or a blood derived sample is used), the sample used foranalysis will preferably be cell-free (e.g. serum or plasma).Alternatively faeces may be used.

The sample may be treated prior to being used in the assay method of theinvention. Thus the sample may be treated to remove any cells and/or anysample components not being assayed for. The sample may also be treatedto concentrate or dilute the sample or to transfer the calprotectin to aseparate medium, e.g. solid substrate. For example the sample may bediluted by adding a buffer or other aqueous medium. Alternatively, asample, particularly a plasma or serum sample, may be used directly.

The sample is optionally treated with calcium or a calcium mimic (e.g.ions of another alkaline earth metal), prior to being used in the assaymethod of the invention. The calcium or calcium mimic may be any formwhich provides Ca²⁺ ions (e.g. CaCl₂). If calcium is used thenpreferably sufficient calcium or calcium mimic is added to the sample tosaturate the calcium binding sites of calprotectin. For example, a tenmolar excess of calcium source may be added, more preferably, a fivemolar excess or especially preferably a three molar excess.

While assays for calprotectin are known and may be used in the method ofthe invention, there has not previously been any suggestion thatcalprotectin is a marker or indicator of potential for CVD or propensityto CVD. In other words, there has not previously been any suggestionthat the calprotectin concentration of symptom-free subjects might beused as a marker or indicator of potential for or propensity to CVD and,in particular, there has not been any suggestion that calprotectinconcentration might be used as the marker or indicator in an assaymethod suitable for mass screening of healthy (i.e. symptom-free)subjects.

Any known assay method for calprotectin may be used in the assay methodof the invention. Thus, for example, the method disclosed in U.S. Pat.No. 4,833,074 (Fagerhol et al.) for the isolation of calprotectin andfor the subsequent production of monospecific anti-sera thereto may beused to produce anti-calprotectin antibodies for use in any conventionalassay method. The anti-calprotectin antibodies produced may be used, forinstance, in enzyme linked- and radio-immunoassays.

A NycoCard® (Axis-Shield PoC, Oslo, Norway) immunoassay format forcalprotectin may also, for example, be used. This assay uses a solidphase, sandwich-format in which the test device comprises a membranecoated with immobilised anti-calprotectin antibodies. Thus the sample(optionally diluted) is applied to the device and when the sample flowsthrough the membrane, any calprotectin present is captured. Thecalprotectin immobilised on the membrane is then treated with agold-antibody conjugate which binds to the calprotectin-antibody complexand the intensity of colour (due to the gold beads), as determined byabsorbance of red light, is proportional to the amount of calprotectin.The concentration of calprotectin can therefore be calculated from acalibration curve prepared in the conventional manner.

Alternatively, the commercial test (Calprest®) for calprotectin, forexample, in faeces (available from) Eurospital® may be used. This assayuses a polyclonal antibody against calprotectin in an enzyme linkedimmuno-sorbent assay system. Thus calprotectin present in a sample takenfrom a subject becomes bound to antibody, which is adsorbed to thesurface of a plastic well. A substrate for the enzyme is then added andthe intensity of the coloured product produced is proportional to theamount of enzyme and therefore to the amount of calprotectin. Theconcentration of calprotectin can therefore be calculated from acalibration curve prepared in the conventional manner.

Indeed, both mono- and polyclonal anti-calprotectin antibodies areavailable commercially. Egg and rabbit polyclonals are, for example,available from Norwegian Antibodies AS and Axis-Shield Diagnosticsrespectively, whilst mouse monoclonal antibody may be obtained from DakoA/S, Denmark. Any anti-calprotectin antibody obtained, for example, byany conventional technique for making antibodies, may be used in themethod of the invention. For instance, rabbit anticalprotectin antibodyas well as monoclonal antibodies can be produced according to theprotocol described in Harlow and Lane (1988), Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, New York, N.Y.

Alternatively, anti-calprotectin antibodies may be prepared by regularlyinjecting a calprotectin-containing solution into chickens, and thencollecting the yolks of the chicken's eggs. Chicken egg polyclonalantibody can then be isolated according to conventional techniques andpurified by affinity chromatography.

Preferably, the assay method of the invention is used for mass screeningof healthy (e.g. CVD symptom-free) subjects. Where potential for CVD orpropensity to CVD is detected, the subject may be subjected to furthertesting (e.g. using more expensive techniques specific to CVD) toconfirm the presence or absence of CVD.

In general, besides the sample under evaluation, calibration sampleswith known calprotectin content will also be assessed in the performanceof the assay method. Such determinations can be used to plot acalibration curve from which the calprotectin content of the sampleunder investigation may be determined. The nature of the calibrationsamples and selection of conversion or adjustment factors used in thedetermination of the calprotectin may vary depending, for example, onthe manner in which the calprotectin is detected in the assay techniqueactually used and on other aspects of the method which affect the assayresult, for example, buffer composition, assay conditions etc.

Typically calibration samples having calprotectin contents of 0 to 5000mg/L will be used. The reference range within which the value forcalprotectin concentration will generally be found is 0.1 to 10 mg/L.

In general, the concentration of calprotectin in the serum and plasma ofhumans with little or no potential for CVD or propensity to CVD will bein the range 0.01-0.75 mg/L. More specifically, the concentration ofcalprotectin in the serum and plasma of such humans will be in the range0.05-0.70 mg/L, even more specifically 0.10-0.66 mg/L, for instance, inthe range 0.15-0.45 mg/L. For example, the concentration of calprotectinin the serum or plasma of a female with little or no potential for CVDor propensity to CVD will be in the range 0.09-0.53 mg/L, for example,about 0.31 mg/L or 0.30 mg/L. The concentration of calprotectin in theserum or plasma of a male with little or no potential for CVD orpropensity to CVD will be in the range 0.12-0.66 mg/L, for example,0.30-0.39 mg/L, especially 0.31 mg/L.

A calprotectin concentration in serum or plasma of greater than 0.75mg/L will generally be very strongly indicative of potential for CVD orpropensity to CVD. Thus a threshold value above which the assay may beheld to be predictive of CVD potential or propensity to CVD maygenerally be in the range 0.32-0.77 mg/L, for example, about 0.67 mg/L,preferably about 0.70 mg/L, especially about 0.76 mg/L. More preferablythe threshold value above which the assay may be held to be predictiveof potential for or propensity to CVD is in the range, 0.30-0.50 mg/L,even more preferably in the range 0.32-0.47 mg/L, for example about 0.45mg/L.

In general, the concentration of calprotectin in the faeces of humanswith little or no potential for CVD or propensity to CVD will be in therange 0.01-10 mg/L. More specifically, the concentration of calprotectinin the faeces of such humans will be in the range 0.05-9.0 mg/L, evenmore specifically 0.50-8.0 mg/L.

A calprotectin concentration in faeces of greater than 10 mg/L willgenerally be strongly indicative of potential for CVD or propensity toCVD. Thus a threshold value above which the assay may be held to bepredictive of CVD potential or propensity to CVD may generally be about9 mg/L, more preferably about 10.5 mg/L, especially about 11 mg/L.

However, the threshold values are better calculated from calprotectindeterminations using the same assay techniques for the same body fluidsample type from a range of patients of similar type (age, sex, weight,species etc.) from healthy through early stage CVD to serious CVD. Evenmore preferably, the threshold values will be values determined for thesame patient at an earlier, healthy stage. Thus, particularly at-risk,individuals could monitor their calprotectin levels on a routine basis(e.g. every 6-months to 1 year) in mass screening programmes.

In a preferred assay method of the present invention, said methodfurther comprises additionally assessing the concentration of anothermarker for potential to CVD in the sample taken from the subject.Examples of suitable markers may be homocysteine, activated factor XII,cholesterol, cholesterol:HDL ratio, fibrinogen, tissue-type plasminogenactivator, Factors V, VII and VIII, lipoprotein (a), von Willebrandfactor antigen, plasmin-α2 antiplasmin complex, prothrombin fragment1+2, thrombin-antithrombin III complex, fibrinopeptide A, fibrindegradation products, D-dimer, activated protein C-resistance, factorVIIc and VIIa, thrombin, serum amyloid A, vascular adhesion moleculesand coronary calcium. Preferably the second marker is selected fromhomocysteine or C-reactive protein.

More preferably the assay method of the present invention furthercomprises additionally assessing the concentration of C-reactive protein(CRP) in the sample taken from the subject. Preferably, theconcentration of CRP is assessed simultaneously or sequentially to saidcalprotectin assay.

The measurement of CRP may be effected using any standard immunoassaytechnique (e.g. ELISA, RIA etc.) or may be determined by NycoCard®(available from Axis-Shield PoC, Oslo, Norway).

A calprotectin concentration in serum or plasma of greater than 0.75mg/L in addition to a CRP concentration of greater than 1.75 mg/L willgenerally be very strongly indicative of potential for CVD or propensityto CVD. Preferably, the presence of the above-mentioned concentrationsis more strongly indicative of potential for CVD or propensity to CVDthan a calprotectin or CRP concentration alone.

Thus threshold values of calprotectin and CRP above which the assay maybe held to be highly predictive of CVD potential or propensity to CVDmay generally be 0.32-0.77 mg/L and 1.70 mg/L respectively, morepreferably about 0.67 mg/L and 1.75 mg/L respectively, still morepreferably about 0.70 mg/L and 2.00 mg/L respectively, especially about0.76 mg/L and 2.25 mg/L respectively. More preferably the thresholdvalues above which the assay may be held to be predictive of potentialfor or propensity to CVD are in the range, 0.30-0.50 mg/L and 0.75 mg/Lrespectively, even more preferably in the range 0.32-0.47 mg/L and 0.75mg/L respectively, for example about 0.45 mg/L and 0.75 mg/Lrespectively.

Viewed from a further aspect, the present invention provides an assaykit for use in the method of the invention, said kit comprising reagentsand instructions for the performance of the assay method and for theinterpretation of the results and, optionally, calprotectin-containingreference samples, and optionally, a detector. Preferably, said assaykit further comprises the reagents and instructions for determination ofCRP concentration.

The instructions in the kit may for example be in the form of a label, amanual or an instruction leaflet; however, they may instead take theform of a computer program or a data carrier, e.g. a computer disk.

The detector, where present, will generally be one capable of detectinga reporter species, e.g. a spectrometer, a nuclear radiation detector, ascattered light detector, etc.

The reagents will be agents suitable for calprotectin determination,e.g. suitable reagents are specified in the literature associated withthe available tests for calprotectin such as from Eurospital® andNycoCard (available from Axis Shield ASA, Oslo, Norway) cited herein.The reagents mentioned in U.S. Pat. No. 4,833,074 may also be suitable.

A particularly preferred assay method for assessing the concentration ofcalprotectin in the present invention is a particle-based immunoassay.This is a sensitive technique which is based on turbidimetricdetermination of the calprotectin concentration. The sensitivityprovided by the assay advantageously allows for the relatively lowconcentrations of calprotectin in body fluid (e.g. plasma or serum)samples to be determined with a high level of precision. At the sametime, relatively high concentrations of calprotectin can also bemeasured with accuracy.

Turbidimetric determination also has the advantage that no solid surfaceis required for physical separation in the assay and numerous washingand/or separation steps are not required. Thus compared to prior arttechniques (e.g. ELISA), the homogenous turbidmimetric determination ofcalprotectin is quick and easy to perform and may, for instance, beautomated. Compared to automation of non-homogeneous techniquesinvolving, for example a solid surface, automation of a turbidimetricbased assay is relatively facile. Also, the resulting automatedhomogenous process is often more reliable being less prone, for example,to break down.

An automated turbidimetric assay is also fast, allowing for a highthroughput of samples, and is relatively cheap to run. Typically it canbe performed using a commercially available robot, e.g. the Cobas Miraor Hitachi 711, both of which are available from Roche Diagnostics. Suchan automated assay is particularly attractive when routine testing ofindividuals for potential for or propensity to CVD is envisaged.

For turbidimetric determination of calprotectin concentration, thecalprotectin-containing sample will generally be a body fluid, e.g.urine, cerebrospinal fluid, oral fluid, synovial fluid or empyema fluid,or more preferably, blood or a blood derived sample. When this is thecase, the sample used for analysis will preferably be cell-free (e.g.serum or plasma)

Thus the sample may be treated to remove any cells and/or any samplecomponents not being assayed for. The sample may also be treated toconcentrate or dilute the sample or to transfer the calprotectin to aseparate medium, e.g. solid substrate. For instance, the sample may bediluted by adding water, a buffer or other aqueous medium.Alternatively, a sample, particularly a serum or plasma sample, may beused directly.

The sample is optionally treated with calcium or a calcium mimic, priorto being used in the assay method. The calcium or calcium mimic may beany form which provides Ca²⁺ ions (e.g. CaCl₂). If calcium is used thenpreferably, sufficient calcium or calcium mimic is added to the sampleto saturate the calcium binding sites of calprotectin. For example, aten molar excess of calcium source may be added, more preferably, a fivemolar excess or especially preferably a three molar excess.

Opacity, for turbidimetric determination of calprotectin concentration,will generally be generated by contacting the calprotectin-containingsample, or an aliquot thereof, with an anti-calprotectin antibody,antibody fragment or mixture of anti-calprotectin antibodies (e.g. amixture of monoclonal antibodies). The egg polyclonal anti-humancalprotectin antibody commercially available from Norwegian AntibodiesAS may, for example, be used to generate opacity. Any anti-calprotectinantibody obtained, for example, by any conventional technique for makingantibodies, may be used in the method of the invention.

The antibodies, or antibody fragments, which are used for turbidimetricdetermination of calprotectin concentration preferably show no or littlecross reactions with other blood proteins that may be present in theeluate. The quantity of antibody, or antibody fragment, used should ofcourse be optimised against calprotectin-containing standard samples asopacification arises from the hook effect whereby multiple calprotectinbinding generates the opacification centres. Calprotectin, as mentionedabove has numerous antibody binding sites, and is particularly suitablefor detection in such an assay.

In one preferred embodiment, the anti-calprotectin antibody, or antibodyfragment, may be immobilised by binding or coupling, either directly orindirectly, to any well known solid support or matrix which is commonlyused for immobilisation. Preferably the solid support or matrix takesthe form of particles, preferably nanoparticles. Conveniently the solidsupport may be made of glass, silica, latex, metal (e.g. gold) or apolymeric material (e.g. polyethylene). Preferably the solid support ismade of a polymeric material such as polyethylene.

Binding or immobilisation of the anti-calprotectin antibody or antibodyfragment may be achieved using any conventional technique. For example,avidin (available from Pierce Chemical Company) may be immobilised onchloromethyl activated polystyrene nanoparticles (available fromInterfacial Dynamic Corporation, US) by agitation in buffer (e.g. atroom temperature for 24 hours) and then used in conjunction with biotinlabelled anti-calprotectin antibodies (prepared according toconventional techniques in the art). Thus, for example, plasma takenfrom the subject to be tested for potential for, or propensity to, CVDis added to a solution of avidin-coated nanoparticles in a quartzcuvette of a spectrophotometer, followed by the addition of biotinlabelled anti-calprotectin antibody. Turbidimetric readings are thentaken.

Alternatively, the biotin labelled antibodies may be added prior to theaddition of plasma or serum. In other words, whilst the same reagentsare typically used regardless of the instrument used for turbiditydetection, the precise sequence in which the various reagents are addedmay vary. Generally, the sequence used should be in accordance with theinstructions accompanying the spectrophotometer used (e.g. a ShimadzuUV-160 spectrophotometer).

Turbidimetric readings are made (i.e. the light absorption at a suitablewavelength is measured at regular intervals) and the light absorptionrelative to a reference is determined. Optionally, multiple wavelengthinstruments may be used to make turbidimetric readings and may providemore precise results. Suitable instruments for taking turbidimetricreadings include the Cobas Mira, Roche Integra and Merck's Turbiquant.

In an alternative experimental set-up, the anti-calprotectin antibody,or antibody fragment, may be immobilised directly on chloromethylactivated nanoparticles (available from Interfacial Dynamic Corporation,US). For instance, anti-calprotectin antibody (e.g. the egg polyclonalantibody available from Norwegian Antibodies AS) may be mixed with theabove-mentioned activated particles in a buffer (10 mM borate, 15 mMsodium chloride, pH 9.0) and agitated (e.g. at room temperature for 24hours) to furnish anti-calprotectin antibody-coated nanoparticles. Suchnanoparticles may be used for turbidimetric determination ofcalprotectin concentration by adding them to a sample of plasma orserum, taken from the subject to be tested for potential for, orpropensity to CVD, in a buffer and taking turbidimetric readings inkinetic mode.

Alternatively, the plasma or serum may be added to the anti-calprotectinantibody-coated nanoparticles. In other words, whilst the same reagentsare typically used regardless of the instrument used for turbiditydetection, the precise sequence in which the various reagents are addedmay vary. Generally, the sequence used should be in accordance with theinstructions accompanying the spectrophotometer used (e.g. a ShimadzuUV-160 spectrophotometer).

Examples of automated robots which are suitable for taking turbidimetricreadings in accordance with the assay method of the invention includethe Cobas Mira and Hitachi 711, both of which are available from RocheDiagnostics.

The particles to which the antibody, or antibody fragment, may be boundare typically spherical. The size of the particles used in the assay mayeffect the precision with which the calprotectin concentration ismeasured. Whilst larger particles allow for lower concentrations ofcalprotectin to be detected, their reduced surface area means that theyhave a lower binding capacity. For example, doubling the particlediameter, halves the binding capacity of a mass unit of particles.

Additionally increasing the particle diameter increases the level ofbackground light absorbance and light suspension at the wavelengthstypically used in such assays (e.g. 330 to 600 nm). Thus whilst largerparticles increase the sensitivity of the assay, this may be accompaniedby some loss of accuracy and in particular, an increase in the number offalse negative results obtained. This is particularly likely to be thecase with samples containing relatively high calprotectin concentrations(i.e. those samples obtained from individuals with a high potential foror propensity to CVD) wherein the nanoparticle-bound binding sites maybecome saturated without all of the calprotectin becoming bound.

These counter-acting effects associated with changing the particle size(e.g. increasing the particle size increases sensitivity but decreasesaccuracy) represents a significant problem to be overcome in thedevelopment of a sensitive assay for detecting the range of levels ofcalprotectin present in body fluids.

Also it is preferable in the assay method of the invention that theparticles used allow for a wide range of calprotectin concentrations tobe determined with precision. This may mean that a high level ofconfidence can be attributed to both a negative result (i.e. aconcentration falling below the threshold value) as well as a positiveresult. It is particularly preferred in the assay method of theinvention that samples having a calprotectin concentration in the range0.5-50 mg/L (e.g. 1-40 mg/L) can be measured.

The particles to which the antibody, or antibody fragment, may be boundare typically spherical with a diameter of 1-150 nm, for example 10-90nm or 15-60 nm, for instance, 44 nm. In a particularly preferred assaymethod of the invention the particles to which the antibody or antibodyfragments are bound have a diameter of 55-140 nm, more preferably 65-110nm, for example, 70-90 nm.

Alternatively the diameter of the particles can be measured onceantibodies or antibody fragments are bound to their surface. In thiscase, the diameter of the antibody or antibody fragment coated particlesis preferably 65-140 nm, more preferably 75-120 nm, still morepreferably 80-100 nm. Coated particles of these sizes are especiallypreferred when the sample tested is plasma.

The particles, in both the “nude” and coated states, preferably have adiameter which does not itself enable absorption of the wavelength oflight used for spectrophotometric determination. Thus the suspension ofcoated nanoparticles is approximately (e.g. substantially) transparentuntil calprotectin induced aggregate formation occurs resulting in theformation of aggregates having a larger diameter. Such aggregates havethe ability to absorb the wavelength of light used by thespectrophotometer.

Further, the particles are preferably substantially all of the samesize, more specifically all of the same diameter. Preferably,monodisperse metal (e.g. gold) or polymer particles are used.Monodisperse polymer particles are available from Dynal Biotech AS,Oslo, Norway.

Whilst not wishing to be bound by theory, it may be that the use ofimmobilised antibody or antibody fragments increases the sensitivity ofthe assay by increasing the size of any calprotectin derived opacitygenerating sites and therefore the amount of light scattered therefrom.By using a solid support or matrix (e.g. nanoparticles) which issubstantially all of the same size it may be that the sensitivity of theturbidimetry assay is further increased.

As is routine in turbidimetric assays, a polymeric opacificationenhancer, such as polyethyleneglycol, is preferably also added to theeluate.

Before the turbidimetric determination is made, the fraction, antibodyor antibody fragment, preferably bound to a nanoparticle, and optionallyenhancer may be incubated for a short period, e.g. 5 minutes to an hour,preferably about 10 minutes, at room temperature. Optionally, indetermining calprotectin concentration using the turbidimetry technique,a kinetic reading mode may be used.

The light used in the determination of opacification should have anappropriate wavelength, for example, 300-600 nm. In this regard it wasfound that use of a 300-450 nm filter, preferably a 340 nm or a 405 nmfilter, furnished particularly good results. Use of a 560 nm filter mayalso yield especially good results.

In general, in addition to the sample under evaluation calibrationsamples with known calprotectin contents will also be assessed in theperformance of the assay method. Such determinations can be used to plota calibration curve from which the calprotectin content of the sampleunder evaluation can be determined. Preferably calibration sampleshaving calprotectin contents of up to 5000 mg/L (e.g. 1500, 1000, 750,250, 100) or up to 100 mg/L (e.g. 75, 50, 25, 5, 1.0 and 0.5 mg/L) willbe used. More preferably the calibration samples have a calprotectincontent of up to 10 mg/L (e.g. 10, 8, 6, 4, 2 mg/L), still morepreferably up to 5 mg/L (5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5 mg/L).

The above described turbidimetric assay for the determination ofcalprotectin is surprisingly reliable, quick, cheap, facile and amenableto automation. This is in contrast to the currently available assaymethods which are relatively complex and are not directly applicable tothe automated multi-task diagnostic machines commonly used by diagnosticlaboratories.

Automation is particularly desirable where numerous mixing, additionand/or dilution steps are involved since these may be achieved with ahigher degree of accuracy. Robots may also offer a higher level ofreliability and/or reproducability. Automation also increasesthroughput.

The currently available assay methods which may offer reasonable levelsof precision (e.g. ELISA) are, however, difficult to automate. This isat least in part because they typically involve numerous washing andseparation steps (e.g. attachment to a solid surface) and automation ofnon-homogeneous processes is often problematic. Also these processestypically involve a relatively large number of steps which increases thecomplexity of any automated protocol. Other conventional techniques fordetermination of calprotectin (e.g. nephelometry) offer high precisionbut require special equipment to carry out the necessary measurements.Specialised equipment is not typically easy to incorporate into anautomated protocol.

Indeed there is a continuing need for cheap, reliable, quick and facilecalprotectin assays for use in diagnostic techniques.

Thus, according to a further aspect, the present invention provides anassay method for the determination of calprotectin in acalprotectin-containing body fluid, said method comprising the steps of:

(a) obtaining a calprotectin-containing liquid sample of, or derivedfrom, said fluid;

(b) contacting said sample of said body fluid with an, optionallynanoparticle-bound, anti-calprotectin antibody or antibody fragment, tobind said calprotectin;

(c) optionally, adding an opacity enhancer; and

(d) assessing the calprotectin content by turbidimetry.

Such as assay may be useful in the diagnosis of various diseaseconditions which are characterised by abnormal levels (e.g. high levels)of calprotectin. Such disease conditions include: rheumatic diseases(e.g. rheumatoid arthritis, juvenile rheumatoid arthritis, systemiclupus erythematosus), Sjøgrens syndrome, intraocular inflammatoryconditions, cystic fibrosis, acute and chronic lung disease, lungcarcinoma (squamous cells), pulmonary cancers, colorectal cancer,inflammatory bowel disease, gastric cancer, colorectal adenoma orcancer, Chrohn's disease, ulcerative colitis, gastrointestinal mucosalinflammation, urinary stones, alcoholic liver disease, oral inflammatorymucosal disease, CNS inflammatory disease (e.g. multiple sclerosis andacute encephalitis), HIV infection, secondary CNS infections in HIVinfected patients, urinary tract infections, cystitis, pyelonephritis,endogenous posterior uveitis, haematological conditions (e.g.leukaemia), febrile conditions (infectious and non-infectious), acutemyocardial infarction and apheresis.

Thus viewed from a further aspect the invention provides a method ofdiagnosis of any of the above-mentioned diseases, comprising the methodas described hereinbefore followed by comparison of said calprotectincontent with a predetermined threshold value. The threshold valueindicative of any particular disease state may be determined by anyconventional method known in the art. Preferably the method is used forthe diagnosis of CVD.

A body sample used in the turbidimetric assay method may be anycalprotectin-containing sample, e.g. a body fluid or tissue sample, or asuspension etc. Preferably, the sample will be a body fluid, e.g. urine,cerebrospinal fluid, oral fluid, synovial fluid or empyema fluid, ormore preferably, blood or a blood derived sample. When this is the case(i.e. blood or blood derived sample is used), the sample used foranalysis will preferably be cell-free (e.g. serum or plasma).Alternatively faeces may be used.

Preferably, the body sample is selected to provide the most sensitiveindication of the disease being diagnosed. Thus whilst blood, plasma orserum might be tested to diagnose infections (e.g. HIV, bacterialinfection), rheumatic disease, leukaemia etc., faeces might be testedduring diagnosis of diseases associated with the gastrointestinal tract(e.g. Crohn's disease, ulcerative colitis, colorectal cancers).

Viewed from yet a further aspect, the invention provides a kit for adiagnostic turbidimetric assay according to the invention, said kitcomprising:

preferably, a calprotectin solution of known concentration and morepreferably a set of such solutions having a range of calprotectinconcentrations;

one or more anti-calprotectin antibodies or antibody fragments,optionally immobilised on a solid support (e.g. nanoparticles);

preferably, a light transmitting vessel;

preferably, an opacification enhancer; and

preferably, a detector.

If desired an automated apparatus may be arranged to receive acalprotectin-containing body fluid sample, apply the anti-calprotectinantibody or antibody fragment, optionally immobilised on a solid support(e.g. nanoparticles), optionally apply an opacification enhancer, andassess calprotectin content. Such an apparatus is also deemed to fallwithin the scope of the invention.

The invention will now be described further with reference to thefollowing non-limiting Examples and the accompanying figures in which:

FIG. 1 is the distribution curve for calprotectin in the 200 subjectstested. The summary statistics for FIG. 1 are:

Anderson-Darling Normality Test A²: 8.160 P-value: 0.000 Mean 0.403Standard Deviation 0.238 Variance 0.057 Skewness 1.679 Kurtosis 3.302 N199 Minimum 0.070 1st Quartile 0.240 Median 0.340 3rd Quartile 0.500Maximum 1.370 95% confidence limit for Mu 0.370 0.436 95% confidencelimit for Sigma 0.217 0.264 95% confidence limit for Median 0.310 0.370

FIG. 2 is the distribution curve for calcium score in the 200 subjectstested. The summary statistics for FIG. 2 are:

Anderson-Darling Normality Test A²: 25.037 P-value: 0.000 Mean 217.060Standard Deviation 399.000 Variance 159201 Skewness 3.462 Kurtosis15.341 N 200 Minimum 0.00 1st Quartile 0.00 Median 78.00 3rd Quartile259.25 Maximum 2794.00 95% confidence limit for Mu 161.42 272.70 95%confidence limit for Sigma 363.35 442.46 95% confidence limit for Median0.00 117.33

FIG. 3 is the distribution curve for hsCRP in the 200 subjects tested.The summary statistics for FIG. 3 are:

Anderson-Darling Normality Test A²: 21.221 P-value: 0.000 Mean 2.331Standard Deviation 2.970 Variance 8.819 Skewness 2.243 Kurtosis 5.103 N197 Minimum 0.150 1st Quartile 0.540 Median 1.150 3rd Quartile 2.580Maximum 16.30 95% confidence limit for Mu 1.913 2.748 95% confidencelimit for Sigma 2.703 3.296 95% confidence limit for Median 0.933 1.375

FIG. 4 is the distribution curve for homocysteine in the 200 subjectstested. The summary statistics for FIG. 4 are:

Anderson-Darling Normality Test A²: 2.535 P-value: 0.000 Mean 8.391Standard Deviation 1.966 Variance 3.867 Skewness 0.875 Kurtosis 0.730 N199 Minimum 5.100 1st Quartile 7.100 Median 8.100 3rd Quartile 9.600Maximum 15.300 95% confidence limit for Mu 8.117 8.666 95% confidencelimit for Sigma 1.790 2.181 95% confidence limit for Median 7.620 8.400

FIG. 5 is a dot plot for the distribution of calprotectin between thecalcium positive and calcium negative results; and

FIGS. 6 and 7 are the ROC curves for calprotectin and hsCRP for all 200subjects tested and for the male subjects tested respectively.

EXAMPLE 1: ANTI-CALPROTECTIN ANTIBODY (a) Isolation of Calprotectin

Calprotectin may be isolated according to the methods described inExamples 1 and 2 of U.S. Pat. No. 4,833,074 (Fagerhol).

Calprotectin may alternatively be purified from human buffy coats. Acell-suspension in 2.5 mM EDTA is made by the addition of EDTA (50 mM,pH 7) to cells. The cells are then washed in 160 mM ammonium chloride/10mM sodium hydrogen carbonate for 3 minutes and centrifuged (160×g) for10 minutes at 4° C. The resulting pellet is washed in EDTA (2.5 mM)/NaCl(150 mM) and centrifuged (55×g) for a further 10 minutes at 4° C. Thepellet is then resuspended in 0.625 mM EDTA/18.75 mM Diemal, pH 7.4 andfrozen at −70° C. for at least 24 hours.

Following thawing, the resulting material is centrifuged (at 3700×g) for30 minutes, then the supernatant is removed and filtered (with a 0.45 μmfilter available from Millipore), then loaded onto a DEAE(diethylaminoethyl) Sepharose ion-exchange column (available fromPharmacia), pre-prepared using a binding buffer (e.g. 0.63 mM EDTA/18.75mM Diemal, pH 7.4). Any non-binding material passes through the columnand is eluted. Once all of the non-binding material is eluted from thecolumn, pure calprotectin is eluted using a calcium-containing elutionbuffer (e.g. 75 mM Diemal buffer/10 mM CaCl₂). About 25 mg calprotectinis obtained per buffy coat.

(b) Preparation of Anti-Calprotectin Antibody

Anti-calprotectin antibodies may be prepared according to the methoddescribed in Example 3 of U.S. Pat. No. 4,833,074 (Fagerhol)

Chicken egg polyclonals may alternatively be prepared. A solutioncomprising calprotectin (0.5 mg/ml) and Freund's adjuvant is injectedinto chickens every 14 days four times (or for two months), and thenonce every 1 month. After 12 weeks, the eggs of thecalprotectin-injected chicken may be collected and their yolks removed(without the film). Following dilution in HCl (5 mM), the yolk iscentrifuged and the supernatant is collected. The supernatant is thenfiltered and treated with saturated ammonium sulfate to a finalconcentration of 3.8 M. The mixture is centrifuged and the precipitateproduced is collected and dissolved in buffer (0.11 M sodium acetate,0.15 M NaCl, pH 7.4). The resulting solution is finally dialysed with amembrane having a pore size of 10,000 kD and then purified by affinitychromatography.

The column typically used for affinity chromatography comprises anactivated matrix of succinimide-activated sepharose (HiTrap NHSactivated available from Amersham-Pharmacia) which is suitable for theimmobilisation of calprotectin. More specifically, the activated resinreacts spontaneously, at pH 7-8, with free amines in the calprotectin.For chromatography the dialysis solution is usually diluted to aconcentration of about 3 mg/ml in PBS prior to its application to thecolumn. The anti-calprotectin antibodies are subsequently eluted using 6M urea in ice cold PBS or 0.1 M sodium citrate solution, pH 3.0.Preferably, 0.1 M sodium citrate solution is used. Following elution,the anti-calprotectin antibody containing fractions are immediatelydiluted and dialysed in PBS.

EXAMPLE 2: TURBIDIMETRIC ASSAY FOR CALPROTECTIN (a) Preparation ofAvidin-Coated Nanoparticles

600 μm of 4.2% w/v chloromethyl activated nanoparticles (diameter 44 nm)available from Interfacial Dynamic Corporation, US are dialysed againstwater with a membrane having a pore size of 10,000 kD. 0.5 ml of aborate (10 mM) and sodium chloride (15 mM) solution at pH 9.0 is addedand mixed. 10 mg avidin, dissolved in 0.5 ml of a 10 mM borate and 15 mMNaCl solution at pH 9 (available from Pierce Chemical Company) is addedand the mixture is agitated at room temperature for 24 hours. 40 μl ofglycine solution (2M, pH 9.0) is then added and the mixture is agitatedfor a further 4 hours at room temperature.

The particles are then diluted to a volume of 100 ml and diafiltrated,firstly in 500 ml of a 10 mM borate and 15 mM sodium chloride solutionat pH 9.0 and secondly in a 25 mM Tris, 150 mM sodium chloride and 0.01%Tween® 20 solution at pH 7.4 (available from Sigma US) using a PelliconXL Filter (cut off 300,000) and a labscale TTF System (available fromMillipore) in accordance with the instructions supplied from theinstruments suppliers. The desired concentration of avidin-coatednanoparticles is finally obtained by centrifugation and re-suspension ofthe particles in a 25 mM TRIS, 150 mM sodium chloride and 0.01% Tween®20 solution. Any aggregates formed during this preparation procedure maybe removed by slow centrifugation.

(b) Assay for Calprotectin Using Avidin-Coated Nanoparticles

A suspension having a concentration of about 0.30 mg particles of theabove-described avidin-coated nanoparticles per ml is prepared bycentrifugation and re-suspension of the above-described preparation in a25 mM TRIS, 150 mM NaCl, 0.1% Tween® 20 and 2% PEG 6000 solution at pH7.4 (available from Sigma). 500 μl of this particle suspension is mixedwith a plasma sample (about 20 μl), taken from a subject being testedfor propensity to CVD, in a reading quartz cuvette of a recordingspectrophotometer (e.g. a Shimadzu UV-160). The absorption of 340 nmmonochromatic light is recorded and after 60 s, 75 μg ofanti-calprotectin antibody labelled with 0.15 nmol biotin (e.g. biotinlabelled affinity purified egg polyclonal purchased from NorwegianAntibodies AS, Norway), diluted in 50 μl of a 25 mM TRIS, 150 mM NaCland 0.1% Tween® 20 solution at pH 7.4 is added to the quartz cuvette andmixed. The absorption of 340 nm monochromatic light is immediatelyrecorded using a reference cuvette containing a solution of 25 mM TRIS,150 mM NaCl and 0.1% Tween® 20 at pH 7.4, and again at regular intervals(e.g. every 2 minutes) until about 15 minutes has elapsed. The increasein absorption at each time point is calculated in accordance withstandard turbidimetric reading in kinetic mode or “end-point” readings.That is, the increase in light absorption at each time-point iscalculated relative to the reading made prior to the addition ofantibody-coated nanoparticles and/or at the end of the recording.

A calibration curve is constructed by carrying out an identicalprocedure with standards having a known concentration of calprotectin.The concentration of calprotectin in the sample can then be calculatedfrom the calibration curve.

EXAMPLE 3: ALTERNATIVE TURBIDIMETRIC ASSAY FOR CALPROTECTIN (a)Preparation of Anti-Calprotectin Antibody Coated Nanoparticles

1 ml of 4.2% w/v chloromethyl activated nanoparticles (diameter 44 nm)available from Interfacial Dynamic Corporation, US are dialysed againstwater with a membrane having a pore size of 10,000 kD. 0.5 ml of a 10 mMborate and 15 mM sodium chloride solution at pH 9.0 is then added. 27 mgof purified anti-calprotectin antibodies (e.g. affinity purified eggpolyclonal antibodies available from Norwegian Antibodies AS, Norway)are dialysed against a 10 mM borate and 15 mM sodium chloride solutionat pH 9.0.

Following addition of the nanoparticles to the purifiedanti-calprotectin antibodies the mixture is agitated for 24 hours atroom temperature. 40 μl of a glycine solution (2 M at pH 9.0) is thenadded and the mixture is agitated for a further 4 hours at roomtemperature.

The particles are then diluted to total volume of 100 ml anddiafiltrated against 1000 ml of a 10 mM borate and 15 mM sodium chloridesolution at pH 9.0 to which 0.1% Tween® 20 and 3 mg/ml egg albumin isadded using a Pellicon XL filter (cut of 300,000) and a labscale TFFsystem (available from Millipore) in accordance with the instructionssupplied from the instruments suppliers. The desired concentration ofanti-calprotectin antibody-coated nanoparticles is finally obtained bycentrifugation and re-suspension of the particles in solution. Anyaggregates formed during this preparation procedure may be removed byslow centrifugation.

(b) Assay for Calprotectin Using Anti-Calprotectin Antibody-CoatedNanoparticles

A suspension comprising 400 μg of the above-described antibody-coatednanoparticles in 50 μl of a 10 mM borate, 15 mM NaCl, 0.1% Tween® 20, 3g/l egg albumin solution at pH 9.0 is prepared.

Simultaneously, 20 μl of plasma, taken from the subject being tested forpotential for CVD, in 500 μl assay buffer (25 mM TRIS, 150 mM NaCl, 0.1%Tween® 20 and 2% PEG 6000 at pH 7.4 (available from Sigma) is put in areading quartz cuvette of a recording spectrophotometer (e.g. ShimadzuUV-160) and the light absorption of 340 nm monochromatic light ismeasured. After 60 s, the above-mentioned suspension comprising 400 μgof antibody-coated nanoparticles is added, and mixed in the cuvette. Thelight absorption immediately after adding the antibody-coatednanoparticles is recorded, and again at regular intervals (e.g. every 2minutes) until about 15 minutes has elapsed. The increase in lightabsorption at each time-point is calculated relative to the reading madeprior to the addition of antibody-coated nanoparticles and/or at the endof the recording. In other words, turbidimetric readings in kinetic modeor “end-point” readings are made.

A calibration curve is also constructed by carrying out an identicalprocedure with standards having a known concentration of calprotectin.The concentration of calprotectin in the sample can then be calculatedfrom the curve.

EXAMPLE 4: TURBIDIMETRIC ASSAY FOR CALPROTECTIN (a) Preparation ofStreptavidin-Coated Nanoparticles

600 μm of 4.2% w/v chloromethyl activated nanoparticles (diameter 67 nm)available from Interfacial Dynamic Corporation, US are dialysed againstwater with a membrane having a pore size of 10,000 kD. 0.5 ml of aphosphate (10 mM) and sodium chloride (150 mM) buffer solution at pH 7.4is added together with 10 mg streptavidin, dissolved in 0.5 ml of a 10mM phosphate and 150 mM NaCl buffer solution at pH 7.4 (available fromPierce Chemical Company) and the mixture is agitated at room temperaturefor 24 hours. 40 μl of glycine solution (2M, pH 9.0) is then added andthe mixture is agitated for a further 4 hours at room temperature.

The particles are then diluted to a volume of 100 ml and diafiltrated,firstly in 500 ml of a 10 mM borate and 15 mM sodium chloride solutionat pH 9.0 and secondly in a 25 mM Tris, 150 mM sodium chloride and 0.01%Tween® 20 solution at pH 7.4 (available from Sigma US) using a PelliconXL Filter (cut off 300,000) and a labscale TTF System (available fromMillipore) in accordance with the instructions supplied from theinstruments suppliers. The desired concentration of avidin-coatednanoparticles is finally obtained by centrifugation and re-suspension ofthe particles in a 25 mM TRIS, 150 mM sodium chloride and 0.01% Tween®20 solution. Any aggregates formed during this preparation procedure maybe removed by slow centrifugation.

The mean particle size of the streptavidin coated nanoparticles wasmeasured to be 82 nm by Sinteff AS, Norway.

(b) Assay for Calprotectin Using Streptavidin-Coated Nanoparticles

A suspension having a concentration of about 0.60 mg particles of theabove-described avidin-coated nanoparticles per ml is prepared bycentrifugation and re-suspension of the above-described preparation in a25 mM TRIS, 150 mM NaCl, 0.1% Tween® 20 and 1% PEG 6000 solution at pH7.4 (available from Sigma). 500 μl of this particle suspension is mixedwith a plasma sample (about 5 μl), taken from a subject being tested forpropensity to CVD, in a reading quartz cuvette of a recordingspectrophotometer (e.g. a Shimadzu UV-160). The absorption of 560 nmmonochromatic light is recorded and after 60 s, 75 μg ofanti-calprotectin antibody labelled with 0.15 nmol biotin (e.g. biotinlabelled affinity purified egg polyclonal purchased from NorwegianAntibodies AS, Norway), diluted in 50 μl of a 25 mM TRIS, 150 mM NaCland 0.1% Tween® 20 solution at pH 7.4 is added to the quartz cuvette andmixed. The absorption of 340 nm monochromatic light is immediatelyrecorded using a reference cuvette containing a solution of 25 mM TRIS,150 mM NaCl and 0.1% Tween® 20 at pH 7.4, and again at regular intervals(e.g. every 2 minutes) until about 15 minutes has elapsed. The increasein absorption at each time point is calculated in accordance withstandard turbidimetric reading in kinetic mode or “end-point” readings.That is, the increase in light absorption at each time-poi_(n)t iscalculated relative to the reading made prior to the addition ofantibody-coated nanoparticles and/or at the end of the recording.

A calibration curve is constructed by carrying out an identicalprocedure with standards having a known concentration of calprotectin.The concentration of calprotectin in the sample can then be calculatedfrom the calibration curve.

EXAMPLE 5 (a) Preparation of Anti-Calprotectin Antibody CoatedNanoparticles

1 ml of 4.2% w/v chloromethyl activated nanoparticles (mean diameter 67nm) available from Interfacial Dynamic Corporation, US, are dialysedagainst water with a membrane having a pore size of 10.000 kD, and thendiluted to 10 ml with water. 27 mg of purified egg polyclonal antibodies(e.g. affinity purified egg polyclonal antibodies available fromNorwegian Antibodies AS, Norway) are dialysed against a 10 mM borate and15 mM sodium chloride buffer solution and finally diluted to 6 ml in thesame 10 mM borate and 15 mM sodium chloride solution at pH=9.0.

Under agitation, the particles are mixed with the antibodies, andagitation is continued at room temperature for 24 hours. 40 μl of aglycine solution (2 M at pH 9.0) is then added and the mixture isagitated for a further 4 hours at room temperature.

The particles are then diluted to a total volume of 100 ml in 10 mMborate, 15 mM sodium chloride buffer to which 0.1% Tween® 20 and 3 mg/mlegg albumin is added and diafiltrated against 1000 ml of saidborate/sodium chloride buffer to which 0.1% Tween® 20 and 3 mg/ml eggalbumin is added, using a Pellicon XL filter (cut off 300.000 D) and alabscale TFF system (available from Millipore) in accordance with theinstructions supplied from the instrument suppliers, and in the end theparticles are concentrated to a volume of 40 to 100 ml.

The mean diameter of the particles obtained was measured to be 81 nm bySinteff AS, Norway.

(b) Assay for Calprotectin Using Anti-Calprotectin Antibody-CoatedNanoparticles

A suspension of 0.7 mg/ml of the above described anti-calprotectinantibody coated particles is made in 0.25 mM TRIS, 0.15 M NaCl and 0.1%Tween at pH 8.0.

5 μl of a plasma sample, taken from the subject being tested for CVD, isdissolved in assay buffer (460 μl, 25 mM TRIS, 150 mM NaCl, 0.1% Tween,1.0% polyethyleneglycol 6000, pH=7.4) in a reading quartz cuvette in arecording spectrophotometer (e.g. Shimadzu UV-160) and the lightabsorption of 560 nm monochromatic light is measured. After 60 s, 100 μlof the above mentioned suspension comprising 0.7 mg/ml of antibodycoated nanoparticles is added, and mixed in the cuvette. The lightabsorption before and immediately after adding the antibody-coatednanoparticles is recorded, and again at regular intervals (eg. every 20s) until 15 minutes has elapsed. The increase in light absorption ateach time point is calculated relative to the reading made prior to theaddition of antibody coated nanoparticles and at the end of therecording. In other words, turbidimetric readings in kinetic mode and/or“end point” readings are made.

A calibration curve is also constructed by carrying out an identicalprocedure with standards having a known concentrations of calprotectin.The concentration of calprotectin ib the sample can then be calculatedfrom the curve.

EXAMPLE 6: STATISTICAL ANALYSIS Comparison of Calprotectin and OtherMarkers for Detection of Potential for CVD or Propensity to CVD

Coronary calcification has been shown to be strongly associated with theoccurrence of CVD and has also been demonstrated to be a useful methodfor predicting potential for CVD (e.g. mycocardial infarction orstroke).

The extent of coronary calcification is quantitatively measured usedelectron-beam computed tomography (EBCT) and is represented by a calciumscore (CS). A high calcium score represents a high level ofcalcification and a high risk of developing CVD.

In the following study the CS of 200 subjects (100 controls having a CS<100 and 100 cases having a CS >100) aged 45 or greater was tested aswell as their calprotectin, CRP and homocysteine plasma or serum levels.The subjects were either self- or physician-referred asymptomaticindividuals and had had an EBCT scan within the previous 2 years(usually within the previous 6 months) to testing of their plasma orserum for the concentration of calprotectin, CRP and homocysteine.

Methods Calprotectin

Calprotectin was measured by the Calprest® test (distributed byEurospital®, Italy) and data were summarised for the patient and thecontrol group using standard deviation, median, minimum and maximum. 95%confidence intervals for median were calculated. The Anderson-Darlingcalculation was used as a test for normality.

Calprotectin was also summarised by gender using the same summarystatistics.

Calcium Score (CS)

Calcium score was determined by Electron-Beam Computed Tomography (EBCT)scanning. Data for case and control groups were summarised.

High Sensitive C-Reactive Protein (hsCRP)

hsCRP was determined by Dade Behring's “N High Sensitivity CRP” assay(Roberts et al., Clinical Chemistry, 2000, 46:4, p 461-468). Data weresummarised for the patient and the control groups using mean, standarddeviation, median, minimum and maximum.

Homocysteine (Hyc)

Plasma Homocysteine (Hcy) was determined by the Abbott IMx method(Shipchandler, M. T. and Moore E. G., Clinical Chemistry, 1995, 41:7, p.991-994). Data were summarised for the patient and the control groupsusing standard deviation, median, minimum and maximum.

Comparison Between CS and Calprotectin, hsCRP, Hcy

(i) Chi-Squared

The Chi-squared test was used to test for covariance between pairs ofmarkers.

(ii) Odds Ratio

The odds ratio used with the 2×2 cross-tabulation (i.e the chi-squaredtable) is the ratio of the odds of two tests co-varying to the odds oftwo tests disagreeing. Therefore, the odds ratio may be interpreted as ameasure of the magnitude of association between the two tests.

Odds ratio was calculated for CS versus calprotectin, hsCRP and Hcyrespectively.

Comparison of Median Values of the Markers (i) Mann-Whitney Test

This is a non-parametric (the data does not need to be normallydistributed) which was used to test if the median values of two markersare significantly different. Minitab ranks all the data from both setsof data in order, assigning 1 to the lowest up to 200 for the highest.The software then adds up the rank-score for the two groups to becompared and reports the “P” value which indicates the chance thatrandom sampling would result in medians as far apart as that observed inthe experiment.

Calprotectin, hsCRP and Hcy median values were compared to CS split intocase and control groups and the Mann-Whitney test was used to test forsignificance.

(ii) ROC-Curves for Calprotectin and Calcium Score

The ability of a test to discriminate diseased cases from normal casescan be evaluated using Receiver Operating Characteristic (ROC) curveanalysis. ROC curve analysis was used for both calprotectin and hsCRPand as a comparison of the two markers.

The ROC curves were produced using 11 cut-off limits for calculation ofthe sensitivity (y-axis) and the 1-specificity (x-axis). The data fromthe control and the case groups were pooled together and ranked fromlowest to highest value. Analysis was performed on the total populationand on the male population. The areas under the ROC curves werecalculated using the trapezoidal rule.

Results Summary Statistics for Calprotectin

FIG. 1 shows the distribution curve for all 200 subjects tested.

Table 1 below shows the summary statistics for calprotectin in thecontrol and the case groups. The median calprotectin concentration inthe control group is 0.31 mg/L compared to 0.38 mg/L in the case group.The median calprotectin concentration is 0.31 mg/L for the males and0.30 mg/L for the females in the control group. In the case group themedian calprotectin concentration for males is 0.39 mg/L compared to0.31 mg/L for females.

TABLE 1 Summary statistics for Calprotectin Calprotectin Controls CasesAll Females Males All Females Males N 100 59 41 100 15 85 SD 0.228 0.2320.225 0.503 0.202 0.537 95% CI for median 0.28-0.34 0.28-0.33 0.27-0.390.34-0.43 0.19-0.47 0.34-0.45 Median (mg/L) 0.31 0.30 0.31 0.38 0.310.39 Min (mg/L) 0.09 0.09 0.11 0.07 0.08 0.07 Max (mg/L) 1.37 1.37 1.364.85 0.73 4.85

Summary Statistics for Calcium Score

FIG. 2 shows the distribution curve for all 200 subjects tested.

Table 2 below shows the summary statistics for EBCT calcium score in thecontrol and case groups. The median calcium score in the control groupis 0 compared to 259 in the case group. The median calcium score is 0for both females and males in the control group and 315 and 256,respectively, in the case group.

TABLE 2 Summary statistics for Calcium score Calcium score ControlsCases All Females Males All Females Males N 100 59 41 100 15 85 SD 5.60.7 8.7 474.7 365.1 493.3 95% CI for median 0-0 0-0 0-0 313.5-215.0174.8-538.0 300.4-207.5 Median 0 0 0 259 315 256 Min 0 0 0 100 100 100Max 56 5 56 2794 1507 2794Summary Statistics for hsCRP

FIG. 3 shows the distribution curve for all 200 subjects tested.

Table 3 below shows the summary statistics for hsCRP in the control andcase groups. The median hsCRP in both the control group and the casegroup is 1.2 mg/L. The median hsCRP is 1.6 mg/L for females and 0.7 mg/Lfor males in the control group and 1.3 mg/L and 1.1 mg/L, respectively,in the case group.

TABLE 3 Summary statistics for hsCRP hsCRP Controls Cases All FemalesMales All Females Males N 100 59 41 100 15 85 SD 6.63 8.36 1.53 12.703.16 13.71 95% CI for median 0.79-1.47 1.36-2.74 0.56-0.97 0.94-1.560.65-2.53 0.89-1.59 Median (mg/L) 1.17 1.59 0.68 1.23 1.28 1.11 Min(mg/L) 0.15 0.15 0.15 0.17 0.34 0.17 Max (mg/L) 62.1 62.1 9.29 120.011.9 120.0

Summary Statistics for Hcy

FIG. 4 shows the distribution curve for all 200 subjects tested.

Table 4 below shows the summary statistics for Hey in the control andcase groups. The median Hcy concentration in the control group is 7.5μmol/L compared to 8.5 μmol/L in the case group. The median Hcyconcentration is 7.15 μmol/L for females and 8.55 mol/L for males in thecontrol group and 7.35 μmol/L and 8.55 μmol/L respectively in the casegroup.

TABLE 4 Summary statistics for Hcy Hcy Controls Cases All Females MalesAll Females Males N 100 59 41 100 15 85 SD 1.94 1.58 2.18 6.03 2.18 6.4695% CI for 7.7- 8.5 7.1-7.9 8.1-9.5 8.1-10.5 6.8-9.2 8.1-10.9 medianMedian 7.5 7.1 8.5 8.5 7.3 8.5 (μmol/L) Min (μmol/L) 5.3 5.3 5.3 5.1 5.15.2 Max (μmol/L) 14.7 11.8 14.7 65.8 12.4 65.8Comparison Between Calcium Score and Calprotectin, hsCRP, Hcy

(i) Chi-Squared Test

Using Minitab, chi-squared tables were performed (see table 5 below).This test compares expected distributions between sets of data (assuminga random distribution) and those observed. The significance value is ameasure of the degree to which the data is not randomly distributed. Forexample, considering the data for calprotectin positive it would beexpected that there would be an even spilt between calcium positive andnegative (i.e. 30.5 expected in both columns). The observed split,however, is 22 negative and 39 positive indicating a tendency for thecalprotectin positives to co-vary with the calcium positives.

Minitab provides an overall measure of the significance of thesedifferences (both agreement and disagreement) and in this case P is0.009. Thus there is a significant co-variation of calcium withcalprotectin.

TABLE 5 Chi-Square comparison of Calprotectin versus Calcium scoreCalcium (co 100) Calprotectin Negative Positive All (co 0.45 mg/L) (N)(N) (N) Negative Obs 78 61 139 Exp 69.5 69.5 139 Positive Obs 22 39 61Exp 30.5 30.5 61 All Obs 100 100 200 Exp 100 100 200

An analogous comparison was made between calprotectin and hsCRP (seeTable 6 below).

TABLE 6 Chi-Square comparison of Calprotectin versus hsCRP (P = 0) hsCRP(co 1.69 mg/L) Calprotectin Negative Positive All (co 0.45 mg/L) (N) (N)(N) Negative Obs 100 39 139 Exp 88.96 50.04 139 Positive Obs 28 33 61Exp 39.04 21.96 61 All Obs 128 72 200 Exp 128 72 200

(ii) Odds Ratio

Odds ratios can be derived from tables 5 and 6 and give a measure of howfar the values observed deviate from the expected. If the expectedagreement figures are multiplied together and divided by the product ofthe disagreement expected figures a value of 1 should be obtained.

Taking the data in Table 6, for instance, the odds ratio on the expectedvalues is 1: (88.96×21.96)/(50.04×39.04)=1953.64/1953.6=1.

The odds ratio observed in this table is:(100×33)/(39×28)=3300/1092=3.02.

The further the odds ratio from 1 the more pronounced the co-variation;an odds ratio of 3 is significant.

The results of an odds ratio analysis carried out on the co-variancedata obtained from the study are shown in Table 7.

TABLE 7 Summary of Odds-Ratio findings Risk markers Odds ratio (N1#pos,N2#neg) Calcium Calprotectin CRP Calcium Score (co 100) — — — N1 = 100,N2 = 100 Calprotectin (co 0.45 mg/L) 2.27 — — N1 = 61, N2 = 139 CRP (co1.69 mg/L) 1.00 3.02 — N1 = 72, N2 = 128 Hcy (co 12 μmol/L) 1.21 1.321.52 N1 = 11, N2 = 189 (co 10 μmol/L) 1.63 1.55 1.12 N1 = 42, N2 = 158co = cut off

A high odds ratio indicates a high degree of co-variation between thetests. It can be seen from Table 7 that the test for calprotectin givesthe highest odds ratio to calcium score and therefore it can be deducedthat calprotectin has the highest degree of covariance with calciumscore out of calprotectin, CRP and homocysteine.

Additionally, calprotectin gives a high odds ratio with CRP, anothermarker for CVD.

Chi-Squared Analysis Using Minitab

A chi-squared test on the above data, using the same cut-offs as theodds-ratio test, showed significant (P 0.009) agreement between thecalcium score and calprotectin results. In contrast, neither the testfor CRP nor the test for homocysteine showed any significant co-variancewith calcium score.

Significant agreement (P o.ooo) was also found between calprotectin andCRP.

Comparing Median Values (i) Mann-Whitney Test

FIG. 5 shows the dot-plot for the distribution of calprotectin betweenthe calcium positive and the calcium negative results.

The median values for the two groups are 0.310 mg/L for the negativecalcium and 0.375 mg/L for the positive calcium. Mann-Whitney providesthat the sum of the ranks for the negative calcium is 912 and 1108 forcalcium negative and yields a P value of 0.0113.

A Mann-Whitney analysis to test for significant increases in the mediansof each of calprotectin, CRP, and homocysteine concentration in the high(CS >100) and low (CS <100) calcium score groups was also carried out onthe above data. The results showed that the median values forcalprotectin and homocysteine concentration were significantly raised inthe high calcium group (P=0.0112 and 0.0037 respectively) whereas CRPdid not show any significant difference in either group (see Table 8below).

TABLE 8 Comparison of median values of Calprotectin and Hcy Median valuein Median value in low CS group high CS group P value Calprotectin 0.3mg/L 0.4 mg/L 0.0112 Homocysteine 7.5 μmol/L 8.5 μmol/L 0.0037

(ii) ROC Analyses and Curves

FIGS. 6 and 7 shows the ROC curves for calprotectin and hsCRP for allsubjects and the male population. Table 9 below shows the area under theROC curves.

TABLE 9 Area under the ROC curves Group Calprotectin hsCRP All subjects0.604 0.524 Male subjects 0.622 0.502

Agreement Rate Against Calcium Score

The accuracy of each of the calprotectin, CRP and homocysteine (Hcy)tests at the cut-off levels (Hcy co 12 μmol) in Table 7 as a test forpotential to CVD was also assessed assuming that a calcium score >100 isreflective of high risk to CVD.

The results of the analysis are shown in Table 10.

TABLE 10 Calprotectin & Calprotectin Calprotectin Calprotectin & CRPcorrect & CRP incorrect CRP incorrect incorrect & CRP correct correct 5842 25 75 29% 21% 12.5% 37.5% Calprotectin & Calprotectin CalprotectinCalprotectin & Hcy correct & Hcy incorrect Hcy incorrect incorrect & Hcycorrect correct 59 40 24 77 29.5% 20% 12% 38.5%

-   Agreement rate against calcium for calprotectin=58.5% (42+75/200)-   Agreement rate against calcium for CRP=50% (25+75/200)-   Agreement rate against calcium for homocysteine=50.5% (24+77/200)

1-27. (canceled)
 28. A kit for use in a diagnostic assay methodcomprising: one or more anti-calprotectin antibody- or antibodyfragment-coated nanoparticles having a diameter in the range 65-140 nm,wherein said assay method is an assay method for the determination ofcalprotectin in a calprotectin-containing body fluid, said methodcomprising the steps of; (a) obtaining a calprotectin-containing liquidsample of, or derived from, said fluid; (b) contacting said sample ofsaid body fluid with said nanoparticle-bound anti-calprotectin antibodyor antibody fragment, to bind said calprotectin; and (c) assessing thecalprotectin content by turbidimetry.
 29. The kit as claimed in claim 28further comprising a calprotectin solution of known concentration or aset of such solutions having a range of calprotectin concentrations. 30.The kit as claimed in claim 28 further comprising a light transmittingvessel.
 31. The kit as claimed in claim 28 further comprising anopacification enhancer.
 32. The kit as claimed in claim 28 furthercomprising a detector.
 33. The kit as claimed in claim 28, wherein theassay method is a direct turbidimetric assay method.
 34. The kit asclaimed in claim 28, wherein the assay method is a direct turbidimetricassay method for the determination of calprotectin at concentrations of0.5 to 50 mg/L in a calprotectin-containing body fluid.
 35. The kit asclaimed in claim 28 wherein in step a) said liquid sample comprisescalprotectin at a concentration of 0.5 to 50 mg/L.
 36. The kit asclaimed in claim 28 wherein in said assay method increasing opacitycorrelates with increasing calprotectin concentration.
 37. The kit asclaimed in claim 28 wherein said assay is capable of measuringcalprotectin with precision in samples having calprotectinconcentrations throughout the range of 0.5-50 mg/L.
 38. The kit asclaimed in claim 28, wherein said anti-calprotectin antibodies orantibody fragments are polyclonal having a population of calprotectinbinding sites and are immobilised by binding or coupling, eitherdirectly or indirectly, to nanoparticles to form antibody or antibodyfragment coated nanoparticles.
 39. The kit as claimed in claim 37,wherein said anti-calprotectin antibodies or antibody fragments are eggpolyclonal antibodies or fragments thereof.
 40. The kit as claimed inclaim 28, wherein said assay method is a direct turbidimetric assaymethod for the determination of calprotectin at concentrations of 0.5 to50 mg/L in a calprotectin-containing body fluid, said method comprisingthe steps of: (a) obtaining a calprotectin-containing liquid sample of,or derived from, said body fluid, wherein said liquid sample comprisescalprotectin at a concentration of 0.5 to 50 mg/L; (b) contacting saidliquid sample of said body fluid with anti-calprotectin antibodies orantibody fragments, to bind said calprotectin, wherein saidanti-calprotectin antibodies or antibody fragments are egg polyclonalantibodies or fragments thereof having a population of calprotectinbinding sites and are immobilised by binding or coupling, eitherdirectly or indirectly, to nanoparticles to form antibody or antibodyfragment coated nanoparticles; and (c) assessing the calprotectincontent in said contacted liquid sample by turbidimetry, wherein thediameter of the antibody or antibody fragment coated nanoparticles is65-140 nm, wherein increasing opacity correlates with increasingcalprotectin concentration; and wherein said assay is capable ofmeasuring calprotectin with precision in samples having calprotectinconcentrations throughout the range of 0.5-50 mg/L.
 41. The kit asclaimed in claim 28, comprising two reagent mixtures only.